RNA synthesis—phosphoramidites for synthetic RNA in the reverse direction, and application in convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3′-end

ABSTRACT

The present invention relates to novel phosphoramidites, A-n-bz, C-n-bz, C-n-ac, G-n-ac and U are produced with an HPLC purity of greater than 98% and  31 P NMR purity greater than 99%. A novel process of reverse 5′→3′ directed synthesis of RNA oligomers has been developed and disclosed. Using that method demonstrated high quality RNA synthesis with coupling efficiency approaching 99%.

CROSS REFERENCE TO OTHER APPLICATIONS

This application is a continuation application to U.S. application Ser.No. 13/633,857, now U.S. Pat. No. 8,933,214, filed Oct. 2, 2012, whichin turn claims priority to U.S. application Ser. No. 12/584,625, nowU.S. Pat. No. 8,309,707, filed Sep. 8, 2009, which in turn claimspriority to U.S. Provisional Application Ser. No. 61/191,065, filed onSep. 6, 2008. The entire teachings of the above applications areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to the synthesis of novel RNA monomerphosphoramidites, and corresponding solid supports that are suitable fora novel method of RNA oligonucleotide synthesis in reverse 5′→3′direction. This approach leads to very clean oligonucleotide synthesisallowing for introduction of various modifications at the 3′-end cleanlyand efficiently in order to produce high purity and therapeutic gradeRNA oligonucleotides.

BACKGROUND OF THE INVENTION

Defined sequence RNA synthesis in the 3′→5′ direction is now wellestablished and currently in use for synthesis and development of a vastvariety of therapeutic grade RNA aptamers, tRNAs, siRNA and biologicallyactive RNA molecules.

This approach utilizes a ribonucleoside with suitable N-protectinggroup: generally 5′-Protecting group, the most popular beingdimethoxytriphenyl, i.e. the DMT group; 2′-protecting group, out ofwhich most popular is t-Butyldimethylsilyl ether; and, a3′-phosphoramidite, the most popular of which is cyanoethyl diisopropyl(component 1). This component is then coupled with a nucleoside with asuitable N-protecting group, 2′ or 3′ succinate of a ribonucleosideattached to a solid support (component 2). The coupling of component 1and 5′-OH-n-protected-2′,3′-protected-nucleoside (component 3) are alsoachieved in solution phase in presence of an activator leading to dimersand oligoribonucleotides, followed by oxidation (3′→5′ directionsynthesis), also leads to a protected dinucleotide having a3′-5′-internucleotide linkage, Ogilvie, K. K., Can. J. Chem., 58, 2686,1980 (scheme 1).

However, the synthesis of RNA in the reverse direction (5′-3′ direction)has not been achieved so far to the best of our knowledge.

The 2′-silyl ethers of component 1 have been developed extensively andthey are known to have remarkable stability. Solvolysis of silyl ethershave been extensively studied and it is known that bulky alkyl silylethers have a high degree of stability; Bazani, B and Chvalowski, VChemistry of Organosilicon compounds, Vol. 1, Academic Press, New York,1965. Extensive research work was subsequently done by Ogilvie andcoworkers as 2′-hydroxy protecting group for oligo ribonucleotidesynthesis (Ogilvie, K. K., Sadana, K. L, Thompson, E. A., Quilliam, M.A., and Westmore, J. B Tetrahedron Letters, 15, 2861-2864, 1974;Ogilvie, K. K., Beaucage, S. L, Entwistle, D. W., Thompson, E. A.,Quilliam, M. A., and Westmore, J. B. J. Carbohydrate NucleosidesNucleotides, 3, 197-227, 1976; Ogilvie, K. K. Proceedings of the 5thInternational Round Table on Nucleosides, Nucleotides and TheirBiological Applications, Rideout, J. L., Henry, D. W., and Beacham L.M., III, eds., Academic, London, pp. 209-256, 1983).

These studies subsequently led to continued developments of methodswhich were amenable to both solution and solid phase oligonucleotidesynthesis, and the first chemical synthesis of RNA molecules of the sizeand character of tRNA (Usman, N., Ogilvie, K. K., Jiang, M.-Y., andCedergren, R. J. J. Am. Chem. Soc. 109, 7845-7854, 1987; Ogilvie, K. K.,Usman, N., Nicoghosian, K, and Cedergren, R. J. Proc. Natl. Acad. Sci.USA, 85, 5764-5768, 1988; Bratty, J., Wu, T., Nicoghosian, K., Ogilvie,K. K., Perrault, J.-P., Keith, G. and Cedergren, R., FEBS Lett. 269,60-64, 1990). The literature has been amply reviewed in subsequentexcellent publication: Gait, M. J., Pritchard, C. and Slim, G.,Oligonucleotides and Their Analogs: A Practical Approach (Gait, M. J.,ed.), Oxford University Press Oxford, England, pp 25-48, 1991. Otherprotecting groups which have been lately employed for RNA synthesis are:bis (2-acetoxyethyl-oxy) methyl (ACE), Scaringe, S. A., Wincott, F. E.,Caruthers, M. H., J. Am. Chem. Soc., 120: 11820-11821, 1998;triisopropylsilyloxy methyl (TOM), Pitsch, S., Weiss, P. A., Jenny, L.,Stutz, A., Wu, X., Helv. Chim. Acta. 84, 3773-3795, 2001 andt-butyldithiomethyl (DTM) (structure 1), Semenyuk, A., Foldesi, A.,Johansson, T., Estmer-Nilsson, C., Blomgren, P., Brannvall, M.,Kirsebom, L. A., Kwiatkowski, M., J. Am. Chem. Soc., 128: 12356-12357,2006 have been introduced. However, none of these processes is amenableto carry out the synthesis of RNA in reverse direction (5′→3′direction); hence they lack the capability of the convenient andefficient introduction of many ligands and chromophores at the 3′-end ofRNA molecules, achievable through reverse direction synthesis.

Chemically modified RNA have been synthesized having modified arabinosugars, 2′-deoxy-2′-fluoro-beta-D_arabinonucleic acid (FANA; structure2)) and 2′-deoxy-4′-thio-2′-fluoro-beta-D_arabinonucleic acid(4′-Thio-FANA; structure 3) into sequences for siRNA activities, Dowler,T., Bergeron, D., Tedeschi, Anna-Lisa, Paquet, L., Ferrari, N., Damha,M. J., Nucl. Acids Res., 34, 1669-1675, 2006. Amongst the several new2′-protecting groups the chemistry for which have been developed, the2′-protecting 2-cyanoethoxymethyl (CEM) (structure 4) has been shown forproducing very long RNA, however, which also carries out RNA synthesisin the conventional, i.e., the 3′→5′ direction. Furthermore, the qualityof RNA produced by these processes remains in question.

The chemical synthesis of RNA is desirable because it avoids theinefficiencies and limitation of scale of synthesis such as by in vitrotranscription by T7 RNA polymerase, Helm, M., Brule, H., Giege, R.,Florence, C., RNA, 5:618-621, 1999. Chemical synthesis of RNA isdesirable for studies of RNA structure and function, and many usefulmodifications can be achieved selectively, such as site specificintroduction of functional groups; viz., disulphide cross linking as aprobe of RNA tertiary structures, Maglott, E. J., Glick, G. D., Nucl.Acids Res., 26: 1301-1308, 1999.

Synthesis of long RNA is very important for biologically activemolecules such as tRNA, and such synthesis has been achieved; Persson,T., Kutzke, U., Busch, S., Held, R., Harmann, R. K., Bioorgan. Med.Chem., 9:51-56, 2001; Oglvie, K. K., Usman, N., Nicoghosian, K.,Cedrgren, R. J., Proc. Natl. Acad. Sci., USA, 85:5764-5768, 1988;Bratty, J., Wu, T., Nicoghosian, K., Ogilvie, K. K., Perreault, J.-P.,Keith, G., Cedergren, R. J., F.E.B.S. Lett., 269:60-64, 1990;Gasparutto, D., Livache, T., Bazin, H., Duplaa, A. M., Guy, A., Khorlin,A., Molko, D., Roget, A., Teoule, R., Nucl. Acids. Res., 20:5159-5166,1992; Goodwin, J. T., Stanick, W. A., Glick, G. D., J. Org. Chem.,59:7941-7943, 1994. None of the techniques mentioned in this paragraph,however contemplate the synthesis of RNA in reverse direction (5′→3′direction), and hence the practical and convenient introduction of anumber of groups required for selective introduction at 3′-end remainselusive.

The present inventors have observed higher coupling efficiency per stepduring automated oligo synthesis with our reverse RNA amidites,resulting in a greater ability to achieve higher purity and produce verylong oligos. They also demonstrate that the process of this inventionleads to oligonucleotides free of M+1 species, which species lead tocloser impurities as shoulder of desired peak during HPLC analysis orpurification or Gel purification.

The t-butyldimethyl silyl protecting group on 2′-hydroxyl ofribonucleosides has been the group of choice for making3′-phosphoramidites and for utilizing them for oligonucleotide synthesiswhich have been shown to migrate to 3′-hydroxyl position rather easily.This has been documented amply and in detail (Ogilvie, K. K., andEntwistle, D. W. Carbohydrate Res., 89, 203-210, 1981; Wu, T., andOgilvie, K. K. J. Org. Chem., 55, 4717-4734, 1990). Such migrationcomplicates the synthesis of the desired phosphoramidites and requiresan efficient method of purification that clearly resolves correspondingisomers and prevents any contamination of the final monomer.

The present invention is directed towards the synthesis of high purityRNAs, specifically to introduce selected groups at 3′-end ofoligonucleotides of synthetic RNAs. Such RNA's have vast application intherapeutics, diagnostics, drug design and selective inhibition of anRNA sequence within cellular environment, blocking a function ofdifferent types of RNA present inside cell.

Silencing gene expression at mRNA level with nucleic acid basedmolecules is a fascinating approach. Among these RNA interference (RNAi)has become a proven approach which offers great potential for selectivegene inhibition and shows great promise for application in the controland management of various biochemical and pharmacological processes.Early studies by Fire et al., Fire, A., Xu, S., Montgomery, M. K.,Kostas, S. A., Driver, S. E., and Mello, C. C, Nature, 391, 806-811,1998, showed that RNA interference in Caenorhabditis elegans is mediatedby 21 and 22 nucleotide RNA sequences. This was further confirmed as ageneral phenomenon of specific inhibition of gene expression by smalldouble stranded RNA's being mediated by 21 and 22 nucleotide RNA's,Genes Dev., 15, 188-200, 2001. Simultaneous studies by Capie, N. J.,Parrish, S., Imani, F., Fire, A., and Morgan, R. A., confirmed suchphenomenon of specific gene expression by small double stranded (dS)RNAs in invertebrates and vertebrates alike. Subsequently a vast amountof research led to the confirmation of above studies and establishedRNAi as a powerful tool for selective, and very specific gene inhibitionand regulation; Nishikura, K., Cell, 107, 415-418, 2001; Nykanen, A.,Haley, B., Zamore, P. D., Cell, 107, 309-321, 2001; Tuschl, T., Nat.Biotechnol., 20, 446-448, 2002; Mittal, V., Nature Rev., 5, 355-365,2004; Proc. Natl. Acad. Sci. USA, 99, 6047-6052, 2002; Donze, O. &Picard, D., Nucl. Acids. Res., 30, e46, 2002; Sui, G., Soohoo, C., Affarel, B., Gay, F., Shi, Y., Forrester, W. c., and Shi, Y., Proc. Natl.Acad. Sci. USA, 99, 5515-5520, 2002; Paddison, P. J., Caudy, A. A.,Bernstein, E., Hannon, G. J.; and Conklin, D. S., Genes Dev., 16,948-959, 2002.

Besides the natural double stranded (ds) RNA sequences, chemicallymodified RNA have been shown to cause similar or enhanced RNAinterference in mammalian cells using2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid (FANA) into sequences forsiRNA activities, Dowler, T., Bergeron, D., Tedeschi, Anna-Lisa, Paquet,L., Ferrari, N., Damha, M. J., Nucl. Acids Res., 34, 1669-1675, 2006.

Various other modifications to improve siRNA properties have beenpursued, which include alteration in backbone chemistry, 2′-sugarmodifications, and nucleobase modifications, some of which have beenrecently reviewed; Nawrot, B, and Sipa, K., Curr. Top. Med. Chem., 6,913-925, 2006; Manoharan, M. Curr. Opin. Chem. Biol., 8, 570-579, 2004.The PS modifications of siRNA are well tolerated, although some reportsindicate increased toxicity and somewhat reduced efficacy; Harborth, J.,Elbasir, S. M., Vandenburgh, K., Manninga, H., Scaringe, S. A., Weber,K., Tuschl, T., Antisense Nucleic Acid Drug Dev., 13, 83-105, 2003.Among these is also the 2′-Omethyl modification, although it maintains Aform (RNA like) helix, and has been shown to be either retaining orreducing siRNA activity depending on the number of such modificationswithin a sequence, Chiu, Y. L., Rana, T. M., RNA, 9, 1034-1048, 2003. Ithas also been shown that extensive 2′-O-Methyl modification of asequence can be made in the sense strand without loss of siRNA activity,Kraynack, B. A., Baker, B. F., RNA, 12, 163-176, 2006.

Bicyclic locked nucleic acids (LNA's) that confer high binding affinityhave also been introduced in siRNA sequences, especially when thecentral region of siRNA sequence is avoided, Braash, D. A., Jensen, S.,Liu, Y., Kaur, K., Arar, K., White, M. A., and Corey, D. R.,Biochemistry, 42, 7967-7995, 2003. Similarly altritol sugar modifiedoligonucleotides (ANA) have recently been reported. Altrilol sugaroffers a rigid conformation and is shown to form very stable duplexeswith RNA in a sequence specific manner, and further shown to stay in A(RNA type) conformation. It was shown that ANA modified siRNAs targetingMDR1 gene exhibited improved efficacy as compared to unmodifiedcontrols, specifically effective when this modification was near the3′-end of sense or antisense strand; Fisher, M., Abramov, M., Aerschot,A. V., Xu, D., Juliano, R. L., Herdewijn, P., Nucl. Acids Res., 35,1064-1074, 2007.

Among the various requirements for effective RNA interference (RNAi) totake place, a number of observations and facts have been established.Thus the RNA has to be double stranded (ds)) in the region of identityto the target. For the chemical requirement, besides the capability of5′-end to be converted to triphosphates, modifications of A, C, G werefound to be fully compatible with interference activity. Backbonemodification of RNA, such as 2′-fluoro, 2′-amino uracil, 2′-deoxythymidine, and 2′-deoxycytidine appear to stabilize the modified RNA inthe resulting double strand. Amongst the nucleoside base modification5-bromouracil, 4-thiouracil, 5-iodouracil, 5-(3-aminoallyl)-uracil,inosine are readily incorporated in the RNA interference complex (RNAi &RISC complex). Similarly inosine may be substituted for guanosine.

It has been shown that cholesterol-conjugated siRNA can achieve deliveryinto cells and silence gene expression. Further, it has been shown thatlipid conjugated siRNA, bile acids, and long chain fatty acids canmediate siRNA uptake into cells and silence gene expression in vivo.Efficient and selective uptake of siRNA conjugates in tissues isdependent on the maximum association with lipoprotein particles,lipoprotein/receptor interactions and transmembrane protein mediateduptake. It has been shown that high density lipoproteins direct thedelivery of siRNA into liver, gut, kidney and steroidal containingorgans. It has been further shown that LDL directs siRNA primarily tothe liver and that LDL receptor is involved in the delivery of siRNA.These results show great promise for siRNA uptake by an appropriatedelivery system which can be exploited in development of therapeutics.(Article by Marcus Stoffel, OTS, 2007, p. 64.)

It has been proposed that siRNA can be designed with chemicalmodifications to protect against nuclease degradation, abrogateinflammation, reduce off target gene silencing, and thereby improveeffectiveness for target genes. Delivery vehicles or conjugates oflipids and other lipophilic molecules which allow enhanced cellularuptake are essential for therapeutic developments. Such siRNA's arepresently being developed for human target validation, interfering withdiseases pathways and developing new frontier for drug development. AlanSachs, Merck, Oligonucleotide Therapeutics Conference (OTS), page 80,2007.

The 3′-end of sense strand of siRNA can be modified and has been shownto tolerate modification, and that attachment of ligands is most suitedat this end (FIG. 19), as detailed in a number of key publications;siRNA function in RNAi: a chemical modification, Ya-Lin Chiu and TariqRana, RNA, 9, 1034-1048, 2003; M. Manoharan, Curr. Opin. Chem. Biol, 6,570-579, 2004; Nawrot, B. and Sipa, K., Curr. Top. Med. Chem., 6,913-925, 2006; Scaringe, S., Marshall, W. S., Khvorova, A., Naty.Biotechnol., 22, 326-30, 2004.

The introduction of lipophilic or hydrophobic groups and enhancing ofsiRNA delivery and optimization of targets has been addressed andachieved through bio-conjugation (FIG. 19). Generally the attachment isdone, preferably at the 3′-end of senses strand, and occasionally on the3′-end of the antisense strand. The design of nuclease resistant siRNAhas been the subject of intense research and development recently inorder to develop effective therapeutics. Thus base modifications suchas, 2-thiouridine, pseudouridine, dihydrouridine have revealed theeffect on conformations of RNA molecules and the associated biologicalactivity; Sipa, K., Sochacka, E., Kazmierczak-Baranska, J., Maszewska,M., Janicka, M., Nowak, G., Nawrot, B., RNA, 13, 1301-1316, 2007. It wasshown that 2′-modified RNA's especially 2′-Fluoro have great resistancetowards nuclease and are biological active in-vivo, Layzer, J. M.,McCaffrey, A. P., Tanner, A. K., Huang, Z., Kay, M. A., and Sullenger,B. A., RNA, 10, 766-771, 2004. 2′-O-Alkyl-modification, such as2′-Omethyl's and 2′-O-MOE, Prakash, S., Allerson, C V. R., Dande, P.,Vickers, T. A., Siofi, T. A., Jarres, R., Baker, B. F., Swayze, E. E.,Griffey, R. H., and Bhat, B., J. Med. Chem., 48, 4247, 4253, 2005. Thesame authors used 4′-thio modified sugar nucleosides in combination of2′-O alkyl modification for improving siRNA properties and RNAienhancement, Dande, P., Prakas, T. P., Sioufi, N., Gaus, H., Jarres, R.,Berdeja, A., Swayne, E. E., Griffey, R. H., Bhat, B. K, J. Med. Chem.,49, 1624-1634, 2006. The Replacement of internucleotide phosphate withphosphorothioate and boranophosphates of siRNAs show promise in-vivo,Li, Z. Y., Mao, H., Kallick, D. A., and Gorenstein, D. G., Biochem.Biophys. Res. Comm., 329, 1026-1030, 2005; Hall, A. H. S., Wan, J.,Shaughnessy, E. E., Ramsay Shaw, B., Alexander, K. A., Nucl. Acids Res.,32, 5991-6000, 2004.

Bioconjugation of siRNA molecules, biologically RNA molecules, aptamersand synthetic DMNA molecules require, in addition to in vivo stabilityand appropriate modification of nucleosides, a key feature for cellmembrane permeability: Insufficient cross-membrane cellular uptakelimits the utility of siRNA's, other single stranded RNA's or evenvarious DNA molecules. Thus cholesterol attached at 3′-end of siRNA hasbeen shown to improve in-vivo cell trafficking and therapeutic silencingof gene, Soutschek, J., Akine, A., Bramlage, B., Charisse, K., Constein,R., Donoghue, M., Elbasir, S., Geickk, A., Hadwiger, P., Harborth, J.,Nature, 432, 173-0178, 2004.

Among the various conjugations, besides cholesterol, which have beendeveloped are:

-   -   (a) Natural and synthetic protein transduction domains (PTDs),        also called cell permeating peptides (CPPs) or membrane permeant        peptides (MPPs) which are short amino acid sequences that are        able to interact with the plasma membrane. The uptake of        MPP-siRNA conjugates takes place rapidly. Such peptides can be        conjugated preferably to the 3′- of stand strand.    -   (b) Other polycationic molecules can be conjugated at the 3′-end        of either sense or antisense strand of RNA.    -   (c) PEG (polyethylene glycols-oligonucleotide conjugates) have        been used in various complex possess significant gene silencing        effect after uptake in target cells, Oishi, M., Nagasaki, Y.,        Itaka, K., Nishiyama, N., and Kataoka, K., J. Am. Chem. Soc.,        127, 1624-1625, 2005.    -   (d) Aptamers have been used for site-specific delivery of        siRNA's. Since Aptamers have high affinity for their targets,        the conjugates with siRNA act as excellent delivery system,        which result in efficient inhibition of the target gene        expression, Chu, T. C., Twu, K. Y., Ellington, A. D. and Levy,        M., Nucl. Acids Res., 34(10), e73, 2006. These molecules can        once again be conjugated at the 3′-end of siRNA or other        biologically active oligonucleotides.    -   (e) Various lipid conjugations at the 3′-end can be achieved        through the present invention and can be utilized for efficient        internalization of oligonucleotides. The lipophilic moiety can        consist of a hydroxyl function to synthesize a phosphoramidite.        Similarly the lipophilic moiety can have carboxylic function at        the terminus. The later can be coupled to a 3′-amino group        having a spacer, synthesized by last addition of amino linkers        such as C-6 amino linker amidite, of the reverse synthesized        oligonucleotide, to the carboxylic moiety using DCC        (dicyclohexyl cabodiimide) or similar coupling reagent.

This research has been reviewed elegantly by Paula, De. D., Bentley, M.V. L. B., Mahao, R. L., RNA, 13, 431-456, 2007.

Another class of RNA, closely related to siRNA are microRNA, commonlyreferred as miRNA. These are a large class of non coding RNA's whichhave a big role in gene regulation, Bartel, D. P. Cell, 116, 281-297,2004; He, L., Hannon, G. J. Nat. Rev. Genet, 5:522-531, 2004;Lagos-Quintana, M., Rauhut, R., Lendeckel, W., Tuschl, T., Science,204:853-858, 2001. In human genome there are at least 1000 miRNAscattered across the entire genome. A number of these micro RNA's downregulate large number of target mRNAs, Lim, L. P., Lau, N. C.,Garrett-Engele, P., Grimson, A., Schelter, J. M., Castle, J., Bartel, D.P., Linsey, P. S., Johnson, J. M., Nature, 433:769-773, 2005. Differentcombination of miRNAs are possibly involved in the regulation of targetgene in mammalian cell. It has also been shown that siRNA can functionas miRNAs; Krek, A., Grun, D., Poy, M. N., Wolf, R., Rosenberg, L.,Epstein, E. J., MacMenamin, P., da Piedade, I., Gunsalus, K. C.,Stoffel, M., Nat. Genet., 37: 495-500, 2005; Doench, J. G., Petersen, C.P., Sharp, P. A., Genes Dev., 17:438-442, 2003. The miRNA have greatpotential in therapeutics and in gene regulation, Hammond, S. M., TrendsMol. Med. 12:99-101, 2006. A vast amount of effort is being currentlydevoted towards understanding miRNA pathways, their role in developmentand disease, focusing specially on cancer. The miRNA targets are beingdeveloped for therapeutic and diagnostics development. A great number ofmiRNA are being identified and their role is being determined throughmicroarrays, PCR and informatics. Syntheses of RNA designed to targetmiRNA also require RNA synthesis and other modification as required forsiRNA's, for the stability of RNA and the bioconjugation for bettercellular uptakes. The reverse synthesis envisioned by the presentinvention can greatly accelerate the pace of this research anddevelopment.

Synthesis of vast variety of therapeutic grade RNA and siRNA requires amodification or labeling of 3′-end of an oligonucleotide. In the case ofsiRNA, generally it is the 3′-end of sense strand. The synthesis of3′-end modified RNA requiring lipophilic, long chain ligands orchromophores, using 3′→5′ synthesis methodology is challenging, requirescorresponding solid support and generally results in low couplingefficiency and lower purity of the final oligonucleotide, in general,because of the large amount of truncated sequences containing desiredhydrophobic modification.

The present inventors have approached this problem by developing reverseRNA monomer phosphoramidites for RNA synthesis in 5′→3′-direction. Thisarrangement leads to very clean oligonucleotide synthesis allowing forintroduction of various modifications at the 3′-end cleanly andefficiently. In order to enhance stability and eliciting of additionalfavorable biochemical properties, this technique can utilize2′-5′-linked DNA (Structure 5) and RNA (Structure 6) which have beendeveloped in the past.

For the efficient delivery of RNA and to increase cellular concentrationof oligonucleotides, lipids containing oligonucleotides are generallysynthesized, since lipids containing synthetic nucleosides enhanceuptake of many synthetic nucleoside drugs, like AZT. Lipipoid nucleicacids are expected to reduce the hydrophilicity of oligonucleotides.Similarly hydrophobic molecules such as cholesterol can bind to LDLparticles and lipoproteins, and activate a delivery process involvingthese proteins to transport oligonucleotides. It has also been shownthat lipidoic nucleic acids improve the efficacy of oligonucleotides.Shea, R. G., Marsters, J. C., Bischofberger, N., Nucleic Acids Res., 18,3777, 1990; Letsinger, R. L., Zhang, G., Sun, D. K., Ikeuchi, T., Sarin,P. S., Proc. Natl. Acad. Sci. USA 86, 6553, 1989; Oberhauser, B., andWagner, E., Nucleic Acids Res., 20, 533, 1992; Saison, -Behmoaras, T.,Tocque, B., Rey, I., Chassignol, M., Thuong, N. T., Helene, C. The EMBOJournal, 10, 1111, 1991; Reed, M. W., Adams, A. D., Nelson, J. S., MeyeR. B., Jr., Bioconjugate Chem., 2, 217, 1991; Polushin, N. N., Cohen,J., J. Nucleic Acids Res., 22, 5492, 1994; Vu, H., Murphy, M., Riegel,Joyce, M., Jayaraman, K., Nucleosides & Nucleotides, 12, 853, 1993;Marasco, Jr., Angelino, N. J., Paul, B., Dolnick, B. J., TetrahedronLett., 35, 3029, 1994. In the studies, the Tm of a series of hydrophobicgroups, such as adamantane (structure 7), eicosenoic acid (structure 8),and cholesterol were attached to oligodeoxy nucleotide sequences at the3′-end and were hybridized to complementary RNA sequences; the Tm wasfound to be unaffected, which indicates that such groups do notinterfere with oligo hybridization properties; Manoharan, M., Tivel, K.L., Andrade, L. K., Cook, P. D., Tetrahedron Lett., 36, 1995; Manoharan,M., Tivel, K. L., Cook, P. D., Tetrahedron Lett., 36, 3651-3654, 1995;Gerlt, J. A. Nucleases, 2 nd Edition, Linn, S. M., Lloyd, R. S.,Roberts, R. J., Eds. Cold Spring Harbor Laboratory Press, p-10, 1993.

Besides the preceding lipidoic molecules other class of molecules, whichhave shown high promise are short and long chain polyethylene glycols(PEG), Bonora, G. M., Burcovich, B., Veronese, F. M., Plyasunova, O.,Pokrovsky, A. and Zarytova, V., XII International Round TableConference, Nucleosides, Nucleotides and their Biological Applications,La Jolla, Calif., September, 15-19, PPI 89, 1996. For efficient deliveryof synthetic RNA, and DNA molecules PEG attachment to variousoligonucleotides have shown to very favorable properties. PEG-oligomershave shown nice enzymatic stability by preventing fast digestion. Thethermal melting behavior was not affected, thereby still retainingproperties of double strand formation.

The approach of the present inventors to produce reversed RNAphosphoramidites required selective introduction of 5′-esters, such as5′-benzoyl, acetyl, levulinyl or substituted-5′-benzoyl-n-protectedribonucleoside. Subsequent introduction of an appropriate protectinggroup at 2′-position, such as 2′-tBDsilyl or 2′-TOM (triisopropyl silylmethoxy; TOM) was required. In their scheme they show synthesis ofselected molecules to achieve this purpose.

In order to produce target compounds, structures (16a-e), the keyintermediate required was 2′-silylether-5′-O acylated-N-protectedribonucleoside, compounds (23a-e). The first intermediate required forthis was 5′-acylated-N-protected ribonucleoside compound (22a-e). To thebest of our knowledge, the compounds (21a-e) have not been reported. Thecompounds, which are reported for RNA synthesis in the past utilizedintermediates, structure 11, 12 and 13. Various 2′ and 3′ acetates with5′-acetate; viz., 5′-benzoyl protected nucleosides; Reese, B. E.,Jarman, M., Reese, C. B., Tetrahedron, 24, 639, 1968; Neilson, T.,Werstiuk, E. S., Can. J. Chem. 49, 493, 1971; Neilon, T., Wastrodowski,E. V., Werstiuk, E. S., Can. J. Chem., 51, 1068, 1973; Eckstein, F.,Cramer, F., Chem. Ber., 98, 995, 1965; Zemlicka, J., Chladek, S., Tet.Lett., 3057, 1965, Amarnath, V. & Broom, A. D., Chemical Reviews, 77,183-219, 1977. The present invention, however, required free 2′ and 3′hydroxyl groups, such as in structures (22a-e).

In the present invention, comparative synthesis and purification ofRNA's were carried out, both by conventional method (3′→5′) and reverseDirection (5′→3′). Observed were: High Purity of RNAs, Smooth3′-Conjugation-Cholesterol, HEG and PEG (Polyethylene glycols) &Demonstration of Absence of M+1 in Reverse RNA Synthesis

The details of synthesis scheme are outlined in the Scheme (2). The2′,3′-Isopropylidene function is utilized to protect the 2′,3′ hydroxylgroups of ribose of n-protected ribonucleosides. A number of preferredn-protecting groups are shown in the Scheme (2). The 5′-hydroxyl groupis subsequently protected, preferably with benzoyl group to obtaincompounds of general structure 21. The isopropylidene group is thenselectively removed under mild acidic conditions well known in the art.This step leads to compounds of general formula 22. Subsequent reactionwith TBDM Silyl chloride (tert-butyl dimethyl silyl chloride) leads tomono silyl compound of general formula 23. The present inventors haveobserved that 3′-TBDMS group, i.e., the formation of compound structure24 is not preferred in this process. In most of the cases they observeda clean product whose structure was confirmed to be 23 by chemical andanalytical methods.

Purification is carried out at each step either via crystallization orcolumn chromatography at each of the steps of the process mentionedabove. Subsequent reaction with dimethoxytrityl chloride (DMT-chloride)in pyridine leads to 3′-DMT-2′-TBDMS-n-protected nucleosides of thegeneral structure 26. Each of the compounds were fully purified bycolumn chromatography.

Although they utilized TBDMS group to produce 2′-TBDMS ether, othersilyl ethers can be utilized at this step. A careful aqueous/methanolicNaOH hydrolysis resulted in compounds with free 5′-hydroxyl group,general structure 27.

Selective 5′-benzoyl removal with aqueous or methanolic base is wellknown in the art. The compounds 3′-DMT-2′-TBDMS-n-protected nucleosides(structure 27) were purified by silica gel column chromatography. Thepurified compounds (structure 27) were subsequently phosphorylated withphosphorylating reagents, such as n,n-diisopropylamino cyanoethylphosphonamidic chloride or 2-cyanoethyl,n,n,n,n-tetraisopropyl phosphaneto yield the corresponding phosphramidites (structures 16). Both thephosphorylating reagents, n,n-diisopropylamino cyanoethyl phosphonamidicchloride or 2-cyanoethyl, n,n,n,n-tetraisopropyl phosphane are readilyavailable in the market¹, and the methods of phosphorylation to producecorresponding phosphoramidites are well known in the art. ¹ Manufacturedby ChemGenes Corp.

SUMMARY OF THE INVENTION

The present invention provides novel RNA monomer phosphoramidites shownbelow in Structure (14). The synthetic route that has been developedallows obtaining desired phosphoramidites without contamination fromunwanted isomers.

The RNA phosphoramidite monomers in this invention contain 3′-DMT groupin ribonucleosides, carrying 5′-cyanoethylphosphoramidite (CED) andvarious methyloxy or silyl protecting groups at 2′-position of theribose moiety (Structure 14). The solid support has protected RNAnucleosides containing 3′-DMT group and 5′ terminus is attached to solidsupport (Structure 15).

The RNA phosphoramidites can be used in the reverse, 5′→3′-directedoligonucleotide synthesis. A large number of RNA synthesis, Conventional(3′→5′) and Reverse Direction (5′→3′) were performed as part of theexperimentation.

The modification or labeling at 3′-terminus of RNA oligomer can beattached by using corresponding phosphoramidite or active ester at theend of the synthesis and do not require special solid support. Moreover,the approach of this invention leads to a very clean 3′-labeledoligonucleotide and expensive methods of purification are not required.

High Purity levels of RNAs were consistently obtained in the RNAsynthesis by Reverse Direction (5′→3′), resulting in Smooth3′-Conjugation of molecules such as Cholesterol, HEG(hexaethyloxyglycol) and PEG (Polyethylene glycols). It was also furtherdemonstrated that in the RNA synthesis in the reverse Direction (5′→3′),there is absence of M+1 oligonucleotide impurities.

Salient Features of the processes of the present invention, from thediscussion, experimental data and the drawing FIGS. 9-18, describedherebelow, include the following observations:

I. The crude RNA's have much closer impurities (N−1) in the conventionalmethod (3′-5′-direction), as compared to reverse RNA synthesis (5′-3′direction). Therefore after purification RNA synthesized by reverse RNAsynthesis are purer.

II The feature mentioned above is much more visible in the synthesis ofcholesterol attached to 3′-end of RNA (see FIG. 11 vs. FIG. 12).Therefore it is easier to purify RNA with cholesterol at 3′-endsynthesized by reverse RNA synthesis (see FIG. 12).

III. M+1 impurities are essentially absent in the RNA's synthesized byreverse RNA synthesis method. It is postulated that in the molecule,ribonucleoside-3′-DMT-2′-tBDsilyl-5′-phosphoramidites, the 3′-DMT is notcleaved by 5-ethylthiotetrazole or similar activators duringoligonucleotide coupling step and within the coupling time ofoligonucleotide chain extension.

IV. RNA containing macromolecules at the 3′-end which are generallyinaccessible by conventional methods (3′→5′) are easily synthesized byreverse RNA synthesis (5′→3′ direction). These RNA's can be produced inhigh purity.

V. 3′-PEG RNA (21-mer) was synthesized, and after purification wasessentially 100% pure (see FIGS. 16, 17 and 18).

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing will be apparent from the following more particulardescription of example embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingembodiments of the present invention.

FIG. 1. HPLC-Chromatogram ofN⁴-Benzoyl-2′O-TBDMS-3′-O-DMT-adenosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16a), 98.6% purity.

FIG. 2. NMR ³¹P Spectrum ofN⁴-Benzoyl-2′O-TBDMS-3′-O-DMT-adenosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16a), sharp doublet at 149.465 ppm & 149.307 ppm; delta; 0.158, 98.6%purity.

FIG. 3. HPLC-Chromatogram ofN⁴-Acetyl-2′-O-TBDMS-3′-O-DMT-cytidine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16c), 96.9% purity.

FIG. 4. NMR 31P Spectrum ofN⁴-Acetyl-2′-O-TBDMS-3′-O-DMT-cytidine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16c), sharp doublet at 149.447 ppm & 149.197 ppp; delta; 0.250, 100%purity.

FIG. 5. HPLC-Chromatogram ofN²-Isobutyryl-2′-O-TBDMS-3′-O-DMT-guanosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16d), 98.3% purity.

FIG. 6. NMR ³¹P Spectrum ofN²-Isobutyryl-2′-O-TBDMS-3′-O-DMT-guanosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16d), sharp doublet at 149.731 ppm & 149.048 ppm, delta; 0.683, 100%purity.

FIG. 6a . Mass Spectrum of the compound (16 d);N²-Isobutyryl-2′-O-TBDMS-3′-O-DMTguanosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite, Negative ionmass; observed at 969.4; calculated; 970.18.

FIG. 6b . Mass Spectrum of the compound (16 d);N²-Isobutyryl-2′-O-TBDMS-3′-O-DMT-guanosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite,Positive ion mass; observed at 993.0; calculated; 993.18 (+Na).

FIG. 7. Diagrammatic representation of 5′→3′ RNA synthesis andapplication to 3′-modifications.

FIG. 8. Coupling Efficiency Trityl Histogram Notes (21 mer RNAsynthesis): Coupling Efficiency (step wise yield by base leveling):99.6%; Final Yield (by rolling average): 100%; Stepwise Yield formonomer G: 100%; Stepwise Yield for monomer A: 99.2%; Stepwise Yield formonomer C: 99.6%; Stepwise Yield for monomer C: 99.6%; Stepwise Yieldfor monomer U: 100.0%

FIG. 9. Electropherogram of the crude oligonucleotide SEQ ID No: 1 madeby conventional method (3′→5′ direction).

FIG. 9a . Electropherogram of the purified oligonucleotide SEQ ID No: 1made by the conventional method (3′→5′ direction). Expedite Model 8909-1umole scale. Crude purity; 90.78%.

FIG. 10. Electropherogram of the crude 21-mer RNA made by Reverse RNAsynthesis (5′→3′ direction). Expedite Model 8909-1 umole scale. Crudepurity; 78.55. Note: M+1 seems to be non-existent in the reversesynthesis.

FIG. 11. Electropherogram of the crude oligonucleotide SEQ ID No: 2 madeby conventional method. Crude 21-mer RNA with 3′-cholesterol CPG, madeby conventional method (3′→5′ direction). Expedite Model 8909-1 umolescale. Crude purity; 82.83%. Note: The humps present at right side, mostlikely due to M+1 species.

FIG. 12. Electropherogram of the crude oligonucleotide SEQ ID No: 2 madeby Reverse RNA synthesis (5′→3′ direction). Expedite Model 8909-1 umolescale. Crude purity; 85.76%. Note: M+1 seems to be non-existent in thereverse synthesis.

FIG. 13. 21-Mer RNA with 3′-Cholesterol-TEG linker. Reverse direction(5′→3′) synthesis and HPLC purification. 1 umole scale. Purity; 99.9%.

FIG. 14. Electropherogram of the crude oligonucleotide SEQ ID No: 4 madeby reverse RNA synthesis. Crude purity; 56.60%.

FIG. 15. 21-Mer RNA synthesized by reverse direction (5′→3′), followedby HEG (Hexa-ethyloxyglycol) attachment. After HPLC purification,Purity; 94.39%.

FIG. 16. Electropherogram of the crude oligonucleotide SEQ ID No: 3 madeby reverse synthesis. 21-mer RNA synthesis with 3′-PEG(Polyethyleneglycol; MW; 2000). Expedite model 8909-1 umole scalesynthesis. Crude purity; 91.87%. Cleanly separated broad peak present.

FIG. 17. Electropherogram of the oligonucleotide SEQ ID No: 4 made byreverse synthesis. 21-mer RNA synthesis with 3′-PEG (Polyethyleneglycol;MW; 2000). Expedite model 8909-1 umole scale synthesis. Reverse phaseHPLC Purified; Purity; 100%.

FIG. 18. ESI Mass Spectral analysis of 21-mer RNA with 3′-PEG-2000attachment, purified RNA as shown in FIG. 17. The synthesis was carriedout in reverse direction (5′→3′ direction). The PEG-2000 was attached aslast step via the corresponding phosphoramidite, ChemGenes catalog;CLP-3119 Calculated Molecular Weight: 8684.1 Observed Molecular Weight:8681.1 Note: There is a distribution of at least 14 PEG species of theRNA on both sides of the Calculated molecular weight with PEG-2000. Thusspecies from 8417.1 to 8945.3 are present with a molecular weightdifference of a glycol unit (+/−44).

FIG. 19 is an illustration showing that the 3′-end of sense strand ofsiRNA can be modified, and an mRNA that is complementary to theantisense strand.

DETAILED DESCRIPTION OF THE INVENTION

The reverse RNA monomer phosphoramidites in the present invention carrya 3′-DMT group in ribonucleosides, carrying 2′-tert-butyldimethylsilyl(TBDMS)-5′-cyanoethylphosphoramidite (CED) (Structure 16),3′-DMT-2′-TBDMS-5′-succinyl-Icaa CPG-n-protected nucleosides (Structure17) or 3′-DMT-2′-triisopropylsilyloxymethyl (TOM)-5′-CED phosphoramiditegroup (Structure 18).

The invention also teaches the method for preparing the disclosedcompositions. The starting base protected nucleoside 19 affordingisopropylidene protected nucleoside 20. Benzoylation followed byisopropylidene group removal yields 5′-benzoylated nucleoside 22.Consecutive silylation reaction with TBDMS chloride in pyridine providesmixture of 2′- and 3′-TBDMS protected nucleosides (23 and 24) in theratio of 3:2 respectively. After column chromatography isomers have beenresolved and isolated. Further reaction of the isomer 23 afforded3′-DMT-2′-TBDMS protected nucleoside 26.

It is therefore conceivable that during subsequent functionalization of3′-hydroxyl group, there will be significant migration of 2′-tBDsilylgroup

During the functionalization of 3′-hydroxyl group withDMT-(4,4-dimethoxytrityl), no significant migration was observed tooccur. Moreover, the 3′-TBDMS protected isomer 24 also was involved inthe same tritylation reaction as isomer 23 with DMT chloride inpyridine, however nucleoside 25 was not observed in that reaction.Therefore, in case of contamination of the 2′-TBDMS protected nucleoside23 with its isomer 24, unwanted isomer 25 cannot be formed in thetritylation conditions and desired nucleoside 26 can be isolated in highpurity. The 3′-TBDMS protected nucleoside 24 can be utilized in thesynthesis of the desired product and converted into 23 due toisomerization process outlined in Scheme 1.

Removal of 5′-benzoyl group with sodium hydroxide in methanol followedby phosphitylation reaction usingcyanoethyl-N,N′-diisopropylphosphoramidite (CEDP) anddiisopropylethylammonium (DIPA) tetrazolate affords the final reversephosphoramidite 16.

Oligonucleotide synthesis using reverse phosphoramidites was performedin the direction from 5′→3′.

The examples provided below further illustrate the invention; these areillustrative only and should not be construed as in any way limiting thescope of the invention. In particular the following examples demonstratesynthetic methods for obtaining the compounds of the invention. Startingmaterials useful for preparing the compounds of the invention andintermediates thereof, are commercially available or can be preparedfrom commercially available materials using known synthetic methods andreagents. All oligonucleotide sequences are written from the 5′-terminuson the left to the 3′-terminus on the right. The coupling efficiency ofthe 3′-DMT-5′-cyanoethyldiisopropyl (CED) phosphoramidites indicated perstep coupling surpassing 99%, leading to high purity RNA. A large numberof homopolymers and 20-21 mers oligonucleotides have been synthesizedusing these monomer phosphoramidites. The typical data is presented inthe FIG. 8).

Our data show that there is no difference in coupling efficiency duringoligo synthesis using the reverse RNA monomers (for 5′→3′-direction) ascompared to standard 3′-cyanoethyldiisopropyl (CED) phosphoramidites insynthesis in 3′→5′ direction (see FIGS. 9 and 10).

In another embodiment the invention provides method for synthesis ofribonucleic acid oligomers with modification or labeling of 3′-end of anoligonucleotide. The synthesis of 3′-end modified RNA requiringlipophilic, long chain ligands or chromophores fluorophores andquenchers can be performed using corresponding phosphoramidites. Ourdata, as captured in FIGS. 11 and 12, show that 5′→3′-directionsynthesis has very distinct advantage compared to conventional method.

In addition, the 3′-modifications that not available on solid supportsuch as HEG or PEG-2000 can be easily introduced by using5′→3′-direction synthesis and purified by reverse-phase HPLC. Theoligonucleotide SEQ ID No 4 has been purified by RP HPLC affording95-98% pure products (see FIG. 15).

EXPERIMENTAL EXAMPLES Example 1 Synthesis ofN²-Isobutyryl-2′-O-TBDMS-3′-O-DMT-guanosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16d) as shown in the scheme 2N²-Isobutyryl-2′-O-TBDMS-3′-O-DMT-5′-O-benzoyl-guanosine (26d)

To the solution of 4 g (7.0 mmol) of the compound 23d in 60 mL ofpyridine were added 9.5 g (28.0 mmol) of DMT chloride in one portion atroom temperature for 48 hrs. Reaction mixture was quenched with 2 mL ofcold methanol then half of the solvent was removed under diminishedpressure, mixed with 20 mL of chloroform, washed with 50 mL of saturatesodium bicarbonate and 50 mL of brine. Organic layer was separated anddryed over anhydrous Na₂SO₄. Flash chromatography with 5:2:3chloroform/hexanes/acetone provided 1.7 g (27.8%) of the compound 26d.TLC system: 5:2:3 chloroform/hexanes/acetone, Rf=0.42. ESMS 896.1[C₄₈H₅₅N₅O₉Si (M+Na)⁺ requires 896.1].

N²-Isobutyryl-2′-O-TBDMS-3′-O-DMT-guanosine (27d)

To the solution of 14 g (16.0 mmol) of the compound 26d in 196 mL ofpyridine and 21 mL of methanol mixture were added 16 mL of 2 M aqueoussolution of sodium hydroxide (32.0 mmol) dropwise with stirring at 0-5°C. during the course of 25 min. The reaction mixture was neutralizedwith 15 mL of 2 M HCl. The solvent was removed under diminished pressureand residue was extracted with two portions of 25 mL of chloroform.Organic layer was combined, washed with 50 mL of brine and dried overanhydrous Na₂SO₄. Flash chromatography with 5:2:3chloroform/hexanes/acetone provided 10.4 g (84.3%) of the compound 27d.¹H NMR (CDCl₃/D₂O) δ −0.54 (s, 3H), 0.01 (s, 3H), 088 (s, 9H), 1.21 (d,3H, J=7.0 Hz), 1.23 (d, 3H, J=7.0), 2.66 (qq, 1H, J=7.0), 2.89 (d, 1H,J=12 Hz), 3.28 (s, 1H), 3.37 (dd, 1H, J_(5a,5b)=15 Hz, J_(5,4)=2.5 Hz)3.80 (s, 6H), 4.24 (d, 1H, J=5 Hz), 4.83 (dd, 1H, J_(2,1)=8 Hz,J_(2,3)=5 Hz), 5.97 (d, 1H, J=8 Hz), 6.84 (dd, 4H, J=9 Hz, J=2 Hz), 7.23(t, 1H, J=7.5 Hz), 7.30 (t, 2H, J=7 Hz), 7.45 (m, 4H), 7.59 (d, 2H,J=7.5 Hz), 7.77 (s, 1H). ESMS 792.8 [C₄₁H₅₁N₅O₈Si (M+Na)⁺ requires792.9].

N²-Isobutyryl-2′-O-TBDMS-3′-O-DMT-guanosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16d)

To the solution of 10.4 g (13.5 mmol) of the compound 27, 3.4 g (26.12mmol) of ethylthiotetrazole, 4.7 mL (27 mmol) of DIPEA and 1.08 mL (13.5mmol) of N-methylimidazole in 104 mL of acetonitrile were added 8.4 mL(26.12 mmol) of 2-cyanoethyl-N,N,N,N-tetraisopropylphosphane dropwisewith stirring under Ar at room temperature. After 3 hrs the reactionmixture was diluted with 100 mL of ethylacetate and washed with 200 mLof saturated sodium bicarbonate and 200 mL of brine. The organic layerwas separated and dried over 2 g of anhydrous Na₂SO₄. Flashchromatography with 7:2:1 chloroform/hexanes/triethylamine provided 12 g(92.1%) of the compound 16d. ³¹P NMR (CDCl₃) δ 149.05 and 149.73. ESMS993.3 [C₅₀H₆₈N₇O₉PSi (M+Na)⁺ requires 993.2].

Example 2 Synthesis ofN⁴-Acetyl-2′-O-TBDMS-3′-O-DMT-cytidine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16c) N⁴-Acetyl-2′-O-TBDMS-3′-O-DMT-cytidine (27c)

To the solution of 5 g (6.2 mmol) of the compound 26c in 54 mL ofpyridine and 6 mL of methanol mixture were added 6.2 mL of 2 M aqueoussolution of sodium hydroxide (12.4 mmol) dropwise with stirring at 0-5°C. during the course of 25 min. The reaction mixture was neutralizedwith 6 mL of 2 M HCl. The solvent was removed under diminished pressureand residue was extracted with two portions of 15 mL of chloroform.Organic layer was combined, washed with 50 mL of brine and dried overanhydrous Na₂SO₄. Flash chromatography with 5:2:3chloroform/hexanes/acetone provided 4 g (91.9%) of the compound 27c. ¹HNMR (CDCl₃/D₂O) δ 0.07 (s, 3H), 0.16 (s, 3H), 0.97 (s, 9H), 2.24 (s,3H), 3.16 (br.d, 1H, J_(5a,5b)=12 Hz), 3.55 (br.d, 1H, J_(5a,5b)=12 Hz)3.79 (s, 6H), 4.08 (t, 1H, J=4.5 Hz), 4.44 (br.s., 1H), 4.78 (br.s.,1H), 5.61 (d, 1H, J=4.1), 6.80 (dd, 4H, J=7 Hz, J=3 Hz), 7.22 (t, 1H,J=7.5 Hz), 7.27 (t, 2H, J=7 Hz), 7.38 (m, 4H), 7.52 (d, 2H, J=7.3 Hz),8.08 (br.s, 1H). ESMS 724.8 [C₃₈H₄₇N₃O₈Si (M+Na)⁺ requires 724.3].

N⁴-Acetyl-2′-O-TBDMS-3′-O-DMT-cytidine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16c)

Was prepared analogously toN²-Isobutyryl-2′-O-TBDMS-3′-O-DMT-guanosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16d). Yield is 62.6%. ³¹P NMR (CDCl₃) δ 149.1 and 149.5. ESMS 925.0[C₄₇H₆₄N₅O₉PSi (M+Na)⁺ requires 924.4].

Example 3 Synthesis of2′-O-TBDMS-3′-O-DMT-Uridine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16k) 2′-O-TBDMS-3′-O-DMT-Uridine (27k)

To the solution of 30 g (39.3 mmol) of the compound 26k in 450 mL ofpyridine and 45 mL of methanol mixture were added 40 mL of 2 M aqueoussolution of sodium hydroxide (80.0 mmol) dropwise with stirring at 0-5°C. during the course of 25 min. The reaction mixture was neutralizedwith 40 mL of 2 M HCl. The solvent was removed under diminished pressureand residue was extracted with two portions of 50 mL of chloroform.Organic layer was combined, washed with 50 mL of brine and dried overanhydrous Na₂SO₄. Flash chromatography with 6.5:2:1.5chloroform/hexanes/acetone provided 24 g (92.5%) of the compound 9k. ¹HNMR (CDCl₃) δ 0.06 (s, 3H), 0.14 (s, 3H), 0.97 (s, 9H), 2.12 (br.d, 1H,J=4 Hz), 3.16 (br.dd, 1H, J_(5a,5b)=12.7 Hz, J_(5a4)=7.6 Hz), 3.55(br.d, 1H, J_(5a,5b)=12.7 Hz J_(5b4)=4.3 Hz) 3.65-3-64 (m, 1H) 3.79 (s,6H), 4.07 (t, 1H, J=4.3 Hz), 4.28 (t, 1H, J=4.3 Hz), 5.66 (d, 1H, J=4.9Hz), 5.69 (d, 1H, J=8.1 Hz), 6.82 (dd, 4H, J=7 Hz, J=3 Hz), 7.22 (t, 1H,J=7.5 Hz), 7.28 (t, 2H, J=7 Hz), 7.38 (d, 4H, J=8.9 Hz), 7.54 (d, 2H,J=8.8 Hz), 7.68 (d, 1H, J=8.2), 8.74 (br.s, 1H). ESMS [C₃₆H₄₄N₂O₈Si(M+Na)⁺ requires 683.3].

2′-O-TBDMS-3′-O-DMT-Uridine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16k)

To the solution of 24.0 g (36.4 mmol) of the compound 27k, 24 mL (182mmol) of collidine, 2.88 mL of N-methylimidazole in 190 mL of THF wereadded 16.22 mL (72.8 mmol) of 2-cyanoethyl-N,N,diisopropylphosphonamidic chloride dropwise with stirring under Ar atroom temperature. After 1.25 hrs the reaction mixture was diluted with100 mL of ethylacetate and washed with 200 mL of saturated sodiumbicarbonate and 200 mL of brine. The organic layer was separated anddried over 20 g of anhydrous Na₂SO₄. Flash chromatography with 5:4:1ethylacetate/hexanes/triethylamine provided 18 g (80.0%) of the compound16k. ³¹P NMR (CDCl₃) δ 148.9 and 149.6. ESMS 884.1 [C₄₅H₆₁N₄O₉PSi(M+Na)⁺ requires 884.0].

Example 4 Synthesis ofN⁴-Benzoyl-2′O-TBDMS-3′-O-DMT-adenosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16a) N⁴-Benzoyl-2′-O-TBDMS-3′-O-DMT-adenosine (27a)

To the solution of 14 g (15.7 mmol) of the compound 26a in 189 mL ofpyridine and 21 mL of methanol mixture were added 15.7 mL of 2 M aqueoussolution of sodium hydroxide (31.4 mmol) dropwise with stirring at 0-5°C. during the course of 25 min. The reaction mixture was neutralizedwith 12 mL of 2 M HCl. The solvent was removed under diminished pressureand residue was extracted with two portions of 25 mL of chloroform.Organic layer was combined, washed with 50 mL of brine and dried overanhydrous Na₂SO₄. Flash chromatography with 6.5:2:1.5chloroform/hexanes/acetone provided 11.0 g (88.9%) of the compound 27a.Yield is %. ¹H NMR (CDCl₃/H₂O) δ −0.75 (s, 3H), −0.01 (s, 3H), 0.86 (s,9H), 3.03 (t, 1H, J_(5a,5b)=12.8 Hz), 3.29 (s, 1H), 3.47 (br.d, 1H,J_(5a,5b)=12.8 Hz) 3.80 (s, 6H), 4.35 (d, 1H, J=4.9 Hz), 5.17 (dd, 1H,J=8 Hz, J=5 Hz), 5.93 (dd, 1H, J=12 Hz, J=2 Hz), 6.15 (d, 1H, J=8 Hz),6.85 (dd, 4H, J=7 Hz, J=3 Hz), 7.23 (t, 1H, J=7.5 Hz), 7.30 (t, 2H, J=7Hz), 7.48 (m, 4H), 7.52 (t, 2H, J=7.3 Hz), 7.62 (d, 3H, J=7.5 Hz), 8.03(d, 2H, J=7.5 Hz), 8.14 (s, 1H), 8.77 (s, 1H), 9.07 (s, 1H). ESMS[C₄₄H₄₉N₅O₇Si (M+Na)⁺ requires 787.3].

N⁴-Benzoyl-2′-O-TBDMS-3′-O-DMT-adenosine-5′-cyanoethyl-N,N-diisopropyl-phosphoramidite(16a)

To the solution of 11.0 g (12.0 mmol) of the compound 27a, 9.2 mL (60.0mmol) of collidine, 1.1 mL of N-methylimidazole in 88 mL of distilledTHF were added 6.23 mL (24.0 mmol) of 2-cyanoethyl-N,N,diisopropyl-phosphonamidic chloride dropwise with stirring under Ar atroom temperature. After 1.25 hrs the reaction mixture was diluted with50 mL of ethylacetate and washed with 100 mL of saturated sodiumbicarbonate and 100 mL of brine. The organic layer was separated anddried over 10 g of anhydrous Na₂SO₄. Flash chromatography with 5:4:1ethylacetate/hexanes/triethylamine provided 9.0 g 77.7%) of the compound16a. ³¹P NMR (CDCl₃) δ 143.97 and 144.14. ESMS 987.2 [C₅₃H₆₆N₇O₈PSi(M+Na)⁺ requires 987.45].

Comparative ¹H NMR Data of Structures 3′-O-DMT-2′-O-TBDMS Nucleosides27a, 27c, 27d, 27k, 16a, 16c, 16d, 16k and 5′-O-DMT-2′-O-TBDMSNucleosides 28-31

TABLE 1 Comparative ¹H NMR data of structures 27d, 16d and structure 28.Structure H-1′ H-2′ H-3′ H-4′ H-5′ab H-8 CH CH₃ 27d 5.97, d. 4.83, dd4.24, d. 3.28, 3.37, dd. 7.76, s. 2.66, qq. 1.31, d., J = 8.0 J = 8.12 J= 5 br. s. J = 15.0, 2.5 J = 7 J = 7 2.98, d. 1.27, d., J = 15.0 J = 728 5.78, d. 5.13,dd. 4.8, br. s. 4.356- 3.89, dd. 7.84, s. 2.78, qq.1.192, d. J = 7 and J = 7, 5 4.353, m. J = 10, 2.5 J = 7 J = 7 5.72, d.and and 3.54, dd. 1.179, d. J = 6.7 4.87, dd. 4.242- J = 10, 2.5 J = 7 J= 7, 5.5 4.223, m. 3.11 J = 10, 2.5 16d 6.22, d., 8.21, s. J = 7, 5 andand 8.08, s. 5.96, d., J = 5

TABLE 2 Comparative ¹H NMR data of structures 27c, 16c and structure 29.Structure H-1′ H-2′ H-3′ H-4′ H-5′ab H-5 H-6 —COCH₃ 27c 5.61, d. 4.78,4.43, 4.08, t., 3.55, br. d. 6.81, d. 7.21, d. 2.24, s. J = 4.1 br. s.br. s. J = 4.6 J = 12.5 J = 7 J = 7 3.16, br. d. J = 11.5 29 5.90, s.4.34- 4.20- 4.10, 3.58, d.d. 7.12, d. 7.16, 2.25, s. and 436, m. 428, m.br. d. J = 11.5, 2 J = 7 d. 5.62, d. J = 5 3.52, dd. J = 7 J = 5 J =11.5, 2 16c 5.83, d., J = 2 6.32, d., J = 5

TABLE 3 Comparative ¹H NMR data of structures 27k, 16k and structure 30.Structure H-1′ H-2′ H-3′ H-4′ H-5′ab H-5 H-6 27k 5.66, d. 4.28, t. 4.07,t., 3.64-3.65, 3.53, br. dd. 5.68, d. 7.68, d. J = 4.9 J = 4.7 J = 4.3br. m. J =13.7, 4.4 J = 8.1 J = 8.1 3.19, br. dd. J = 13.7, 7.6 30 5.95,d. 4.49, t. 4.05-4.08, 4.10-4.11, 3.49-3.51, m. 5.30, d. 7.95, d. J =2.8 and J = 5 and m. br. s. 3.90-3.93, m. J = 8.2 J = 8.2 5.69, d. 4.19,t. 5.73, d. 7.75, d, J = 4.6 J = 5 J = 8.1 J = 7.2 16k 6.00, d., 7.75,d. J = 4.5 and J = 8.2 6.38, d., 7.92, d. J = 7.1 J = 8.2

TABLE 4 Comparative ¹H NMR data of structures 27a, 16a and structure 31.Structure H-1′ H-2′ H-3′ H-4′ H-5′ab H-2 H-8 27a 6.15, d. 5.17, dd.4.35, d. 3.29, br. s. 3.47, br. dd. 8.14, s. 8.76, s. J = 8 J = 8, 4.8 J= 4.8 J = 11.5, 2 3.04, t. J = 12.3 31 5.82, d. 5.00, t. 4.37- 4.27, dd.3.54, dd. 8.21, s. 8.71, s. J = 7 J = 5.5 4.35, m. J = 7, 3.4 J = 10.7,3 3.40, dd. J = 10.7, 3.8 16a 6.53, d., 8.54, s. 8.87, s. J = 7 and andand 6, 24, d., 8.48, s. 8.79, s. J = 4.5

Example 5 Synthesis of 2′-O-TBDMS-3′-O-DMT-uridine-5′-succinyl-CPG2′-O-TBDMS-3′-O-DMT-Uridine-5′-succinate

To the solution of 2.0 g (3.03 mmol) of the compound 27k and 0.11 g(0.91 mmol) of DMAP in 20 mL of pyridine were added 0.9 g (9.1 mmol) ofsuccinic anhydride with stirring at 37° C. After 12 hrs the reactionmixture was diluted with 30 mL of chloroform and washed with 50 mL ofbrine. The organic layer was separated and dried over anhydrous Na₂SO₄.Flash chromatography with 5:3:2:0.01:0.05chloroform/hexanes/acetone/pyridine/methanol provided 1.8 g (75.9%) of2′-O-TBDMS-3′-0-DMT-uridine-5′-succinate. ¹H NMR (CDCl₃/D₂O) δ 0.05 (s,3H), 0.07 (s, 3H), 0.96 (s, 9H), 2.21 (dt, 1H, J=17 Hz, J=6 Hz),2.40-2.46 (m, 1H), 2.51 (dt, 1H, J=17 Hz, J=6 Hz), 2.60-2.66 (m, 1H),3.42 (t, 1H, J=3.3 Hz), 3.77 (s, 6H), 3.95-3.98 (m, 2H), 4.11 (br.d, 1H,J=12.8 Hz), 4.16 (m, 1H), 5.72 (d, 1H, J=2.8 Hz), 5.74 (d, 1H, J=8.1Hz), 6.79 (dd, 4H, J=9 Hz, J=1.9 Hz), 7.21 (t, 1H, J=7 Hz), 7.25 (t, 2H,J=7 Hz), 7.38 (d, 4H, J=8.9 Hz), 7.48 (d, 2H, J=7.2 Hz), 7.61 (d, 1H,J=8.2 Hz), 7.72 (t, 1H, J=5.9 Hz), 8.61 (br.d, 1H, J=4.4 Hz). ESMS 784.2[C₄₀H₄₈N₂O₁₁Si (M+Na)⁺ requires 783.9].

2′-O-TBDMS-3′-O-DMT-uridine-5′-succinyl-CPG

To the suspension of 18 g of amino-lcca-CPG and 3.6 mL of triethylaminein 60 mL of DMF was added the solution of 1.8 g (2.3 mmol) ofO-TBDMS-3′-O-DMT-uridine-5′-succinate, 0.408 g (3.55 mmol)N-hydroxysuccinimide and 0.586 g (2.76 mmol) of DCC in 4 mL of DMF. Thereaction mixture was warmed to 37° C. After 16 hrs CPG was filtered,washed with 3×20 mL portions of acetonitrile, capped with aceticanhydride in pyridine/N-methylimidazole mixture and washed 3×20 mLportions of acetonitrile. The solid support was dried under diminishedpressure and nucleoside loading was measured by DMT removal procedureyielding 18 g of final product with 44.2 μmol/g loading.

Example 6 Oligonucleotide Synthesis

The following oligonucleotides (Table 5) were synthesized using 3′→5′directed standard RNA phosphoramidite chemistry in 1 μmole scale. Thesyntheses were performed on Expedite 8900 synthesizer using standard RNA1 μmole cycle.

Following synthesis, the controlled pore glass (CPG) solid support wastransferred to a 2 ml microfuge tube. Oligonucleotides were cleaved fromthe CPG and deprotected by incubation for 30 min at 65° C. in 1 ml of40% methylamine solution in water. The supernatant was removed and theCPG was washed with 1 ml of water; supernatants were pooled and dried.The t-butyl-dimethylsilyl protecting group was removed from the RNAresidue by treatment with 250 ul of fresh anhydroustriethylammonium-trihydrogen fluoride at room temperature in ultrasonicbath for 2 hours. The oligonucleotide was precipitated by 1.5 ml ofn-butanol; the sample was cooled at −70° C. for 1 hour then centrifugedat 10,000 g for 10 minutes. The supernatant was decanted, the pellet waswashed with n-butanol one more time.

The oligonucleotides were then purified by reverse-phase HPLC using alinear gradient of acetonitrile in 0.1 M triethyl-ammonium acetate(TEAA) pH 7.2. The entire sample was loaded on a Hamilton PRP-1 column(1.0 cm×25 cm) and eluted with a linear 5% to 50% acetonitrile gradientover 40 minutes. Samples were monitored at 260 nm and peakscorresponding to the desired oligonucleotide species were collected,pooled, and lyophilized.

The oligonucleotide samples were dissolved in 200 ul of sterile waterand precipitated by adding 1 ml of 2% LiClO₄, followed by centrifugingat 10,000 g for 10 minutes. The supernatant was decanted, the pellet waswashed with 10% aqueous acetone.

-   -   The standard dT and cholesterol corresponding solid supports        suitable for oligonucleotide synthesis have been used.

TABLE 5 Oligonucleotide sequences synthesized by  conventional method.SEQ ID  rCrArGrGrUrGrCrArGrArGrCrCrUrUrGrCrCrCTT No: 1 SEQ ID rCrArGrGrUrGrCrArGrArGrCrCrUrUrGrCrCrCTT- No: 2 Cholesterol

-   -   The following oligonucleotides (Table 6) were synthesized using        5′→3′ directed reverse phosphoramidite chemistry in 1 mmole        scale. The same synthesis cycle and ancillary reagents as in        standard process have been used for reverse synthesis. The        reverse rC-lcaa-CPG was used in all oligonucleotide syntheses.        The 3′-modifications of the oligonucleotides SEQ ID No. 2-4 have        been introduced by using cholesterol, PEG-2000 or HEG        phosphoramidites respectively.

TABLE 6 Oligonucleotide sequences synthesized by reverse method. SEQ IDrCrArGrGrUrGrCrArGrArGrCrCrUrUrGrCrCrCTT No: 1 SEQ IDrCrArGrGrUrGrCrArGrArGrCrCrUrUrGrCrCrCTT- No: 2 Cholesterol SEQ IDrCrArGrGrUrGrCrArGrArGrCrCrUrUrGrCrCrCTT- No: 3 PEG/2000 SEQ IDrCrArGrGrUrGrCrArGrArGrCrCrUrUrGrCrCrCTT- No: 4 HEG

Crude oligonucleotides were analyzed by CE and ESI mass-spectrometry.

A vast number of applications are possible for easy attachment at 3′-Endof an oligonucleotide. Some of the examples are outlined in FIG. 7:

1. For attachment of bulky molecules at the 3′-end of the RNA, such ascholesterol, long chain aliphatic chains such as C-18, triethyleneglycols, hexaethylene glycols. Direct coupling with these amidites canbe achieved easily.

2. Attachment of Polyethylene Glycols such as PEG 2000 amidite and PEG4000 amidites at the 3′-end of the RNA molecule.

3. For Easy attachment of 3′-thiol modification. 3′-Disulfides fromreadily available amidites, viz., C-3 disulfide, C-6 disulfide.

4. 3′-Biotin attachment via biotin amidite in a single step and avoidingbiotin CPG for this purpose.

5. Modification of 3′-End of the Sense Strand of siRNA. The modificationof the overhang of the sense strand (3′-End) of siRNA is not expected toaffect targeted mRNA recognition, as the antisense siRNA strand guidestarget recognition. Useful modification for improvement of delivery ofsiRNA can be easily designed.

TABLE 3 Comparative ¹H NMR data of structures 27k, 28k and formula 8.Structure H-1′ H-2′ H-3′ H-4′ H-5′ab H-5 H-6 27k 5.66, d. 4.28, t. 4.07,t., 3.64-3.65, 3.53, br. dd. 5.68, d. 7.68, d. J = 4.9 J = 4.7 J = 4.3br. m. J = 13.7, 4.4 J = 8.1 J = 8.1 3.19, br. dd. J = 13.7, 7.6 Formula8 5.95, d. 4.49, t. 4.05-4.08, 4.10-4.11, 3.49-3.51, m. 5.30, d. 7.95,d. J = 2.8 and J = 5 and m. br. s. 3.90-3.93, m. J = 8.2 J = 8.2 5.69,d. 4.19, t. 5.73, d. 7.75, d, J = 4.6 J = 5 J = 8.1 J = 7.2 28k 6.00,d., 7.75, d. J = 4.5 and J = 8.2 6.38, d., 7.92, d. J = 7.1 J = 8.2

We summarize in the notes below the various innovations, advantages andpossibilities, and some product and process details of the presentinvention. This list is meant to serve as a convenient and illustrativesummary, and is not complete, exhaustive or limiting.

-   -   Derivatized nucleoside and phosphoramidites of general formula        1:

-   -   wherein    -   Y is oxygen or sulfur;    -   W is oxygen, nitrogen, sulfur or fluorine;    -   R₄ is silyl ether such as TBDMS, triisopropylsilyl oxymethylene,        Fmoc, alkyl, aryl, or acetyl, when W is not sulphur; but in case        when W is sulfur R₄ is benzoyl, acetyl or disulfide;    -   Z is DMT, MMT, TMT protecting group;    -   R₁ and R₂ are independently selected from an alkyl or aryl        group;    -   R₃ is cyanoethyl, alkyl or aryl.    -   Derivatized nucleoside attached to solid support of general        formula 2:

-   -   wherein    -   M is a hydrogen radical or Y—CO—;    -   Y is a chain of atoms from 2 to 20 in length, consisting        essentially of a hydrocarbon chain optionally substituted by one        or more heteroatoms independently selected from the group        consisting of oxygen, nitrogen and sulfur, or any linker that is        suitable for linking a solid support thereto, such as CPG,        polystyrene or any other solid support suitable for        oligonucleotide synthesis;    -   W is oxygen, nitrogen, sulfur or fluorine;    -   R is silyl ether such as TBDMS, triisopropylsilyl oxymethylene,        Fmoc, alkyl, aryl, amino or acetyl, when W is not sulphur; but        in the case when W is sulfur R is benzoyl, acetyl or disulfide;    -   Z is DMT, MMT, TMT protecting group.    -   A method for reverse, via 5′ to 3′ direction of oligonucleotide        bond formations shown in formula 10 in synthetic RNA oligomers.        The RNA could consist of natural or modified nucleo bases,        gapmers, phosphodiesters, phosphorothiates, phosphoselenates.        The synthesis may be performed on automated, semi automated        DNA/RNA or other synthesizers or manually. The synthesis can be        performed at various scales from microgram to kilogram scales.

-   -   A method of attachment of modifications to 3′-terminus of RNA        molecules using corresponding phosphoramidites (Formula 11),    -   wherein L is a modification such as biotin or cholesterol, or        selected from the group consisting of fluorophore, quencher        dyes, polyethylene glycols, and peptides.

-   -   Synthesis of automated high purity RNA using Reverse Direction        (5′-3′) RNA synthesis resulting in high purity RNA.    -   3′-Conjugation of RNA with macromolecules such as Cholesterol,        hexaethyloxyglycols (HEG) and Polyethylene glycols (PEG).    -   Application of the automated RNA synthesis in the reverse        Direction (5′→3′), resulting in the absence of M+1        oligonucleotide impurities.    -   The modified nucleosides incorporated by this method mentioned        above could consists of one or more of purine or pyrimidine        modifications, such as but not limited to, 5-fluorouridine,        5-fluorodeoxyuridine, 5-fluorodeoxycytidine, 5-fluorocytidine,        pseudouridine, 5-methyldeoxyuridine, 5-methyluridine,        5-methyldeoxycytidine, 5-methylcytidine, 5-bromodeoxyuridine,        5-bromouridine, 5-bromodeoxycytidine, 5-bromocytidine,        5-iododeoxyuridine, 5-iodouridine, 5-vinyldeoxyuridine,        5-vinyluridine, 5-vinylthymidine, 3-methyldeoxyuridine,        3-methyluridine, 3-methylthymidine, 4-thiouridine,        4-thio-2′-deoxyuridine, 2,6-diaminopurinedeoxyriboside,        3-methylribothymidine, 2,6-diaminopurineriboside,        8-bromo-2′-deoxyadenosine, 8-bromoadenosine,        8-oxodeoxyadenosine, 8-oxoadenosine, 8-oxodeoxyinosine,        8-oxoinosine, 8-bromodeoxyinosine, 8-bromoinosine,        1-methyladenosine, 1-methyl-2′-deoxyadenosine,        1-methyl-2′-deoxyinosine, 1-methyladenosine,        1-methyldeoxyguanosine, 1-methyl-guanosine, ethenoadenosine,        etheno-2′-deoxyadenosine, purine-2′-deoxyriboside,        purine-ribonucleoside, 2-aminopurine-2′-deoxyriboside,        2-aminopurine-ribonucleoside.    -   Labelling of internal positions of an RNA synthesized by this        method is achievable with chromophores such as, but not limited        to Fluoroscein-C-5 dT, Dabcyl-C-5 thymidine, internal carboxyl        group 5-dU-methylacrylate, biotin dT (biotin w attached via        spacer to C-5 of dU), amino-dT (terminal amino attached via C-6        spacer to C-5 dU).    -   The sugar modification of modified nucleosides could consist of        2′-deoxy-2′-fluoro ribo nucleosides (2′-F-ANAs) such as A, C, G,        U, Inosine and modified nucleosides containing 2′-Fluoro, in one        or more positions of an RNA or DNA sequence synthesized by the        method of this invention.    -   The sugar modification of modified nucleosides could consist of        2′-deoxy-2′-methoxy ribo nucleosides (2′-OMe-) such as A, C, G,        U, Inosine and modified nucleosides containing 2′-methoxy, in        one or more positions of an RNA or DNA sequence synthesized by        this method.    -   The sugar modification of modified nucleosides could consist of        2′-deoxy-2′-amino ribo nucleosides (2′-NH2) such as A, C, G, U,        Inosine and modified nucleosides containing 2′-amino, in one or        more positions of an RNA or DNA sequence synthesized by this        method.    -   The sugar modification of modified nucleosides could consist of        2′-deoxy-2′-terminal amino ribo nucleosides (2′-terminal NH2),        attached via spacer from 2-10 atoms on nucleosides such as A, C,        G, U, Inosine and modified nucleosides containing 2′-terminal        amino, in one or more positions of an RNA or DNA sequence        synthesized by this method.    -   The sugar modification of modified nucleosides could consist of        2′-deoxy-2′-methoxy ethoxy ribo nucleosides (2′-MOE), such as A,        C, G, U, Inosine and modified nucleosides containing 2′-MOE, in        one or more positions of an RNA or DNA sequence synthesized by        this method.    -   The sugar modification of modified nucleosides could consist of        other 2′-O-alkyl groups, such as 2′-deoxy-2′-ethoxy, propargyl,        butyne ribo nucleosides (2′-OEt, O-Propargyl, 2′-O-Butyne), such        as A, C, G, U, Inosine and modified nucleosides containing        2′-2′-OEt, 0-Propargyl, 2′-O-Butyne, in one or more positions of        an RNA or DNA sequence synthesized by this method.    -   The sugar modification of modified nucleosides could consist of        2′-deoxy-2′-fluoro arabino nucleosides (2′-F-ANAs) such as A, C,        G, U, Inosine and modified nucleosides containing 2′-F-ANAs), in        one or more positions of an RNA or DNA sequence synthesized by        this method.    -   The sugar modification of modified nucleosides could consist of        2′-deoxy-2′-fluoro 4′-thioarabino nucleosides (4′-S-FANAs) such        as A, C, G, U, Inosine and modified nucleosides containing        4′-S-FANAs in one or more positions of an RNA or DNA sequence        synthesized by this method.    -   The RNA may be carried out with one or more 2′-5′-linkage within        the sequence, at the 3′-end of the sequence or at the 5′-end of        the sequence.    -   The RNA having a 3′-end, may be synthesized by the method of        this invention containing reverse attached deoxy nucleosides        such as dT, dC, dG, thymidine, attached via their 3′-hydroxyl        function.    -   The RNA having a 3′-end may be synthesized by the method of this        invention containing reverse attached ribonucleosides such as        rA, rC, rG, rU, attached via their 2′ or 3′-hydroxyl function.    -   The reverse RNA synthesis may be achieved comprising        2′-triisopropylsilyloxy methyl (TOM) protecting group.    -   The reverse RNA synthesis may be achieved comprising        2′-t-butyldithiomethyl (DTM) protecting group.    -   The reverse RNA synthesis may be achieved comprising the        modified base comprising        2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid (FANA).    -   The reverse RNA synthesis may be achieved comprising the        modified base comprising        4′-thio-2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid        (4′-Thio-FANA).    -   The reverse RNA synthesis may be achieved comprising the        modified sugar using 2′-OMethyl modification.    -   The reverse RNA synthesis may be achieved by using Bicyclic        locked nucleic acids (LNA's).    -   The reverse RNA synthesis may use the modified sugar comprising        altritol sugar modified oligonucleotides (ANA).    -   The reverse RNA synthesis may comprise the step of conjugation        of lipophilic or hydrophobic groups at the 3′-end of the RNA        either through a amidite function on the hydrophobic moiety or        through an amino linker at the 3′-end of reverse synthesized        oligonucleotide having a terminal amino group. The later        synthesis involving a coupling step between amino at the        3′-terminal of oligonucleotide and carboxylic function on the        lipophilic moiety. The lipophilic moieties consist of various        glycol, such as triethylene glycol, hexaethylene glycol,        polyethylene glycols, various lipids.    -   The reverse RNA synthesis may comprise the step of conjugation        of peptides, such as cell penetrating peptides (CPPs) or        membrane permeant peptide (MPPs) utilizing either the free amine        function of such peptides and a 3′-terminal carboxylic function        on the reverse synthesized RNA. The CPPs and MPPs having an        appropriate carboxyl function can be coupled to the free        terminal amino function of a 3′-end of the reverse synthesized        RNA.    -   The reverse RNA synthesis comprise the 2′-5′-linked DNA units or        2′-5′-RNA units within the sequence, at the 3′-end of the        sequence or at the 5′-end of the sequence.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. Any combination ofthe embodiments disclosed in the dependent claims are also contemplatedto be within the scope of the invention.

What is claimed is:
 1. A method for the synthesis of an RNAoligonucleotide, comprising a structure represented by the followingformula:

wherein: B, B′ or B″ is a member selected from the group consisting of9-adeninyl, 1-cytosinyl, 9-guaninyl, 1-uracilyl, 9-inosinyl,5-methyl-1-cytosinyl, 5-methyl-1-uracilyl, 5-fluoro-1-uracilyl,7-deaza-9-adeninyl, and 5-fluoro-1-cytosinyl; n is an integer between 0and 100; L is a nucleoside, a non-nucleoside ligand selected from thegroup consisting of cholesterol attached via a linker or via a spacer,biotin, ethylene glycol, glycerol, a polyethylene glycol, a hexaethyleneglycol, an amino linker, a disulfide linker, a peptide linker, apolypeptide linker, a protein, a fluorophore, a quencher dye, one ormore 2′,5′-linked deoxynucleoside units, one or more 2′,5′-linkedribonucleoside unit, and one or more 2′,5′-linked deoxyribose units,wherein L is attached at the 3′-end of the RNA nucleotide through anintervening phosphate; and the RNA oligonucleotide is synthesized by aprocess in a direction from the 5′-end to the 3′-end, and the processcomprises the steps of: (a) taking a nucleoside solid supportrepresented by Formula 2:

wherein: M is a hydrogen radical or a linker; if M is a linker, then itis represented by the formula Y—C(O) and Y is connected to a solidsupport suitable for oligonucleotide synthesis, wherein Y is ahydrocarbon diradical moiety having a length between 2 carbons and 20carbons, and Y is selected from the group consisting of alkyl, alkenyl,cycloalkyl, aryl, and aralkyl, and the hydrocarbon diradical moietyoptionally consists of intervening —O—, —S—, —S(O)₂— —C(O)— and —NR₆—where R₆ is a hydrogen radical, or a substituted C₁ to C₂₀ alkyl or asubstituted aralkyl; W is selected from the group consisting of anoxygen diradical, an N—H diradical, and a fluorine radical, and R isselected with the proviso that: if W is an oxygen diradical, then R istert butyl dimethyl silyl (TBDMS) or triisopropylsilyl oxymethylene(TOM); and if W is an N—H diradical, then R is of the form R₅X, whereinX is selected from the group consisting of fluorenylmethyloxycarbonyl(Fmoc), trifluoroacetyl, acetyl, alkanoyl and aroyl; and if W is afluorine radical, then R is not present; B is selected from the groupconsisting of nucleoside base radicals consisting of9-(N⁶-benzoyladeninyl)-, 9-(N⁶-acetyladeninyl)-, 9-(N⁶-tert-butylphenoxyacetyladeninyl)-, 9-(N⁶-phenoxyacetyladeninyl)-, 9-(N⁶-isopropylphenoxyacetyladeninyl)-, 1-(N⁴-benzoylcytosinyl)-,1-(N⁴-acetylcytosinyl)-, 1-(N⁴—(N,N-dimethylformamidinyl)cytosinyl)-,1-(N⁴-phenoxyacetylcytosinyl)-,1-(N⁴-tert-butylphenoxyacetylcytosinyl)-, 1-(N⁴-isopropylphenoxyacetylcytosinyl)-, 9-(N²-isobutyrylguaninyl)-, 9-(N²-tert butylphenoxyacetylguaninyl)-, 9-(N²-isopropyl phenoxyacetylguaninyl)-,1-(N⁴-phenoxyacetylcytosinyl)-, 1-(N⁴-tert butylphenoxyacetylcytosinyl)-, 1-(N⁴-isopropyl phenoxyacetylcytosinyl)-, and1-uracilyl-; or B is a modified nucleoside base radical selected fromthe group consisting of 1-(N⁴-benzoyl-5-methylcytosinyl)-,1-(N⁴—(N,N-dimethylformamidinyl)-5-methylcytosinyl)-,1-(N⁴-acetyl-5-methylcytosinyl)-, 1-(5-methyl-uracilyl)-,1-(5-fluoro-uracilyl)-, 1-(N⁴-benzoyl-5-fluorocytosinyl)-,9-(N⁶-benzoyl-7-deazaadeninyl)-,9-(N⁶—(N,N-dimethylformamidinyl)-7-deazaadeninyl)-,9-(N²-isobutyryl-7-deazaguaninyl)-, and9-(N²—(N,N-dimethylformamidinyl)-7-deazaguaninyl)-; Z is a protectinggroup selected from the group consisting of dimethoxy triphenyl (DMT),monomethoxy triphenyl (MMT) and trimethoxy triphenyl (TMT); (b) placinga phosphoramidite represented by Formula 1 in an appropriate containercomponent of an oligonucleotide synthesizer;

wherein Y is an oxygen atom or a sulfur atom; W is selected from thegroup consisting of an oxygen diradical, an N—H diradical, and afluorine radical; and R₄ is selected with the proviso that: if W is anoxygen diradical, then R₄ is tert butyl dimethyl silyl (TBDMS) ortriisopropylsilyl oxymethylene (TOM); and if W is an N—H diradical, thenR₄ is of the form R₅X, wherein X is selected from the group consistingof fluorenylmethyloxycarbonyl (Fmoc), trifluoroacetyl, acetyl, alkanoyland aroyl; and if W is a fluorine radical, then R₄ is not present; B isselected from the group consisting of nucleoside base radicalsconsisting of 9-(N⁶-benzoyladeninyl)-, 9-(N⁶-acetyladeninyl)-,9-(N⁶-tert-butyl phenoxyacetyladeninyl)-, 9-(N⁶-phenoxyacetyladeninyl)-,9-(N⁶-isopropyl phenoxyacetyladeninyl)-, 1-(N⁴-benzoylcytosinyl)-,1-(N⁴-acetylcytosinyl)-, 1-(N⁴—(N,N-dimethylformamidinyl)cytosinyl)-,1-(N⁴-phenoxyacetylcytosinyl)-,1-(N⁴-tert-butylphenoxyacetylcytosinyl)-, 1-(N⁴-isopropylphenoxyacetylcytosinyl)-, 9-(N²-isobutyrylguaninyl)-, 9-(N²-tert butylphenoxyacetylguaninyl)-, 9-(N²-isopropyl phenoxyacetylguaninyl)-,1-(N⁴-phenoxyacetylcytosinyl)-, 1-(N⁴-tert butylphenoxyacetylcytosinyl)-, 1-(N⁴-isopropyl phenoxyacetylcytosinyl)-, and1-uracilyl-; or B is a modified nucleoside base radical selected fromthe group consisting of 1-(N⁴-benzoyl-5-methylcytosinyl)-,1-(N⁴—(N,N-dimethylformamidinyl)-5-methylcytosinyl)-,1-(N⁴-acetyl-5-methylcytosinyl)-, 1-(5-methyl-uracilyl)-,1-(5-fluoro-uracilyl)-, 1-(N⁴-benzoyl-5-fluorocytosinyl)-,9-(N⁶-benzoyl-7-deazaadeninyl)-,9-(N⁶—(N,N-dimethylformamidinyl)-7-deazaadeninyl)-,9-(N²-isobutyryl-7-deazaguaninyl)-, and9-(N²—(N,N-dimethylformamidinyl)-7-deazaguaninyl)-; Z is a protectinggroup selected from the group consisting of dimethoxy triphenyl (DMT),monomethoxy triphenyl (MMT) and trimethoxy triphenyl (TMT); R₁ is analkyl or aryl radical; R₂ is an alkyl or aryl radical; and R₃ iscyanoethyl, alkyl or aryl radical; B is hydrogen or a nucleobase-derivedsubstituent moiety which is optionally functionalized at each primaryamine with an amine protecting group; (c) removing the protecting groupZ from the nucleoside-derived solid support represented by Formula 2 toform a nucleoside with an active OH group at 3′; (d) performing theprocess of RNA synthesis by coupling the nucleoside with the active OHgroup at 3′ of step (c) and the phosphoramidite of Formula 1 in theoligonucleotide synthesizer to result in an oligonucleotide having atleast one protecting group; (e) providing a phosphoramidite with an Lgroup; (f) adding the phosphoramidite with the L group at the end of theoligonucleotide to result in an oligonucleotide having the L group andcontacting the product of L group attachment with an oxidizing agent insufficient quantity to convert all of the P(III) linkages to P(V)linkages; (g) detaching the oligonucleotide having the L group from thesolid support; (h) removing the at least one protecting group from theoligonucleotide; (i) removing a silyl protecting group to result in theoligonucleotide; (j) precipitating the oligonucleotide; and (k)analyzing the oligonucleotide for purity, and the RNA oligonucleotide isessentially free of M+1 species as impurity according to anelectropherogram.
 2. The method according claim 1, wherein L ischolesterol attached via the linker or via the spacer, and n=19.
 3. Themethod according to claim 1, wherein L is polyethylene glycol (PEG), andn=19.